Control of DNA replication is one of the key processes in the regulation of the mammalian cell cycle. Dysregulation of the cell cycle can arise at several stages, for example in the "decision" to begin DNA replication or in the proper completion of the S-phase, and is an important causative factor in a variety of human diseases, the most frequent being cancer. The long term goal of our research is to elucidate in molecular detail the mechanisms that control DNA replication in mammalian cells. Replication of papovaviral DNA in infected cells and in cell-free reactions has proven to be an extremely useful model system toward this end. It has facilitated the identification and characterization of many of the cellular proteins required for replication and has led to a basic understanding of the mechanism of SV40 DNA replication. One aspect of our research program focusses on the role of protein phosphorylation in regulating the activity of several proteins required for the initiation of SV40 viral DNA replication in a cell-free system. For example, phosphorylation of DNA polymerase alpha-primase by cyclin A-dependent kinases inhibits its SV40 origin-dependent replication activity. We are using peptide mapping and MALDI-MS to define the relevant phosphorylation sites, then creating mutations at these sites and characterizing the mutant proteins in viral DNA replication and in a recently devised cell-free human DNA replication system. The possibility that the phosphorylation state of DNA polymerase alpha-primase affects cell cycle progression or genomic stability in vivo is also under investigation. Another open question we plan to address soon is how dephosphorylation of pol a in the cell cycle is controlled.